Effects of Oral Lipid‐Based nutritional supplements on appetite, energy intake, and lipid profile of moderately underweight children

Abstract Oral lipid‐based nutritional supplements (LNS) are designed to ensure dietary adequacy and to improve malnourishment in children. Therefore, this study investigated the effects of 4 weeks of LNS on appetite, energy intake, and lipid profile of moderately underweight children (5–10 years old) with BMI‐Z score between −2 and − 3 SDS, recruited in a single‐blind randomized control trial. In addition to the regular dietary intake, fasting blood samples, anthropometric measurements, energy intake, and appetite responses were obtained before and after 4 weeks of LNS (535 kcal) or PLACEBO (92 kcal). After 4 weeks of supplementation mean energy intake (kcal) (p < .001), body weight (kg) (p < .001), BMI (kg/m2) (p < .01), mid‐upper arm circumference (cm) (p < .01), total cholesterol (mg/dl) (p < .01) and fasting glucose (mg/dl) (p < .01) were raised significantly in the LNS group as compared to the PLACEBO group. No significant changes were detected in appetite responses (p > 0.05). In conclusion, LNS increases the overall energy intake, but does not affect the appetite but may induce hyperglycemia and hyperlipidemia.

partially immunized or not immunized against childhood diseases, making them vulnerable to infections (Imdad et al., 2011) and malnutrition (Hirani, 2012). Poor breastfeeding practices along with low quality of complementary foods also contribute to malnutrition (Mujib et al., 2006).
Among the strategies to revive moderately acute malnourished (MAM) children, the most acceptable regime is the use of LNS (World Health Organization, 2007). A randomized controlled trial conducted by Huybregts et al., observed significant effects on the growth rate of undernourished children after 4 months of LNS (Huybregts et al., 2012). Another study found 29% increase in recovery rate and a significant improvement in weight-for-height in MAM children provided with nutritional supplements (Lazzerini et al., 2013). Similarly, a cluster-randomized study on infants observed that a supplementation of 500 kcal ready-to-use therapeutic food (RTUF) per day decreased the chances of wasting over a follow-up period of 8 months (Isanaka et al., 2009). Similarly, a 4 months′ follow-up non-randomized cohort study on 2238 children observed a positive effect on anthropometric measurements and decreased prevalence of wasting by taking readyto-use supplementary food (RUSF) (Grellety et al., 2012). Similar findings were observed in severely acute malnourished (SAM) and MAM children where the recovery rate was enhanced during supplementation period (Defourny et al., 2009;Karakochuk et al., 2012). In contrast, a study found lower than expected weight gain in underweight children after the intake of LNS (Fatima et al., 2018). Concomitantly, in lean women LNS suppressed the appetite and energy intake after the use of LNS .
Appetite is a physiological process that influences energy intake and is associated with motivational states like hunger, fullness, satiety, and desire to eat (Gibbons et al., 2019). Poor appetite causes insufficient food intake which in turn causes undernutrition in children (Waterlow, 1994). A study found that imbalance in hormones like leptin and insulin may also cause loss of appetite (Arsenault et al., 2007). It was also observed that the use of LNS may reduce the insulin sensitivity . Undernourished people often suffer from dyslipidemia (Lumey et al., 2009) however, no effects on fasting lipids were observed after using LNS for 5 days .
Previous studies have documented the impact of various multimicronutrient supplements on the improvement of appetite in stunted and undernourished children (Dossa et al., 2002;Shakur et al., 2009;Stoltzfus et al., 2004). However, no clinical trial has been conducted to measure the changes in appetite of MAM children.
Since the effectiveness of LNS interventions for the prevention of undernutrition is rapidly increasing (Ashorn et al., 2013), there is noticeable lack of evidence on possible effect of LNS on appetite of MAM children. Many targeted interventional programs have been implemented to improve energy intake in underweight children.
However, the effect of LNS on energy intake and appetite in MAM children is scarce. Similarly, the efficacy of LNS on lipid profile has been demonstrated in many studies in adults and obese children but the data on effects of LNS on lipid profile in MAM children is hitherto unknown. Therefore, this study was designed in the community set-up for school-going MAM children to investigate the effect of LNS on appetite. In addition, changes in glucose, lipid profile, insulin, and energy intake were explored.

| Study design
This single-blind randomized control study was carried out at clinical trial room, IBMS, KMU, and 12 public sector schools and orphanages of Peshawar, from September, 2017 to April, 2018. The study was approved by the Ethical Board of KMU (Study approval No. DIR/KMUEB/EN/000454). Children were approached after gaining permission from schools' administrations. Children with any food or medicine allergies, eating or gastrointestinal disorders were excluded. After taking the anthropometric measurements according to WHO standard procedures, 37 school-going mild-to-moderate underweight children (BMI Z-score between −2 and − 3 SDS) between 5 and 10 years of age were selected. Written as well as verbal informed consent from the guardians (parents) and assent from children were obtained. The children (20 boys and 17 girls) were randomly allocated into two groups using Computer Randomizer software into the LNS group (n = 19) and the PLACEBO group (n = 18). This sample size was calculated using online software "Openepi." The data to calculate this sample size were obtained from a previous study by Fatima et al., . This sample size was enough to detect a difference of approx. 535 kJ of energy intake with 80% power at level of 5% to spot a difference of around two-thirds of the standard deviation between the LNS and the PLACEBO for any measure (Figure 1).
The LNS used in this study was "Acha Mum" by WFP (World Food Programme) providing 535 kcal/day (Table 1). The PLACEBO was prepared with 42 g roasted chickpeas (68 kcal) and 58 g low-fat milk (Nesvita; 24 kcal). It provided 92 kcal/day energy ( Table 1). No conflict of interests was found among any group involved in this study.

| Socio-demographic data
Socio-demographic data were collected using pre-designed proforma. Data regarding parents' characteristics (status, occupation, education, and monthly income), number of siblings and family members, family accommodation/expenditure, and housing quality were recorded from all the participants.

| Main trial days (day 1st and day 31st)
On main trial days, that is, day 1 and day 31, participants reported to the clinical trial room between 07:30 and 08:30 a.m. in fasting state.
After taking fasting blood samples, anthropometric measurements were taken. It included body height, body weight-for-age, skinfold thickness, mid-upper arm circumference (MUAC), and waist/hip ratio.
Participants marked the appetite questionnaires at time point 0, that is, before the consumption of LNS or PLACEBO. The questionnaire was further marked at 30-and 60-minute post supplementation, followed by an ad libitum buffet breakfast at 90 min. The questionnaire was again marked at 120, 150, 180, and 210 min, after the supplementation followed by lunch at 240 min. During the trial, LNS/PLACEBO supplements were provided to children at their school daily for 1 month and empty packets were collected back to check compliance (Figure 2).

| Anthropometric measurements
Height of participants was measured by portable stadiometer using a stretch stature method to the nearest 0.1 cm. Weight was measured by using Beurer digital glass weight scale "GS 200 Allium".
Shakir's measuring tape was used to measure mid-upper arm circumference (MUAC) on the non-dominant hand to the nearest 0.1 cm.
Skin-fold measurements, that is, biceps, triceps, mid-abdomen, and sub-scapular thickness were checked using "Holtain skin-fold caliper" to the nearest 0.2 mm. Waist-to-Hip ratio was obtained by taking the waist and hip measurements using "Lasso-o measuring tape."

| Blood parameters
Blood samples collected in fasting state were analyzed for glucose, insulin, and lipid parameters, that is, total cholesterol F I G U R E 1 Schematic representation of participants' recruitment and follow-up during the trial (TC), triglyceride (TG), high-density lipoproteins (HDL), and lowdensity lipoproteins (LDL). After centrifugation, the blood serum was stored at −80°C. "Roche Cobas Immunoassay E411 Analyzer" was used to analyze insulin and glucose, while "Roche Cobas C501 Chemistry Analyzer" was used for lipid profile analysis.

| Energy intake measurements
Dietary Windiet software 2005 was used to calculate energy intake. To enhance the accuracy of results, three individual researches noted the values and then mean value was taken as final result.

| Multiple pass 24-hour dietary recalls
Dietary data of the participants were collected using multiple pass 24-hour dietary recalls. The data were collected three times for three consecutive days, that is, 3 days before the 1st trial day, 3 days after 1st trial, and 15 days after 1st trial day. This was done to check the effects of supplement on habitual dietary intake of participants. The difference between the regular intake of participants before and after the supplement was also measured and compared.

| Appetite measurement
In this study, five-point Likert scale appetite questionnaires were used to check the responses to assess hunger, satiety, fullness, appetence, and desire to eat. A Likert scale is a psychometric scale used in research that employs questionnaires (Norman, 2010). Each questionnaire included a set of five questions with five responses.
The participants had to choose one response about hunger, satiety, fullness, appetence, and desire to eat. Initially, the questionnaires were marked by the participants in fasted state (at 0 min) and at 30 and 60 min after supplementation followed by breakfast. The questionnaires were marked in pre-lunch period, that is, at 120-, 150-, 180-, and 210-min post-supplementation.

| Hedonic scales measurements
Participants and the parents were inquired separately about the taste and acceptability of LNS using hedonic scales. The parents

F I G U R E 2 First and second trial day procedure
were also asked about any kind of progression noticed by them in their child after 1 month of supplementation.

| Statistical analysis
Data analysis was done using Minitab version 17. To check weightfor-age Z-score (WAZ) score, a software LMS (Least mean squares) Growth was used. Normality of data was checked by Anderson Darling test. Categorical data were compared by Chi-square test.
Appetite questionnaires and lipid profile were checked by paired two samples t-test. p-Value < 0.05 was taken as significant.

| RE SULTS
In this trial, 37 mild-to-moderate underweight children with age range 5-10 years and BMI-Z score between −2 and − 3 SDS were recruited. A total of 32 blood samples (18 boys and 14 girls) were successfully collected by the end of the trial ( Figure 1) and were analyzed for variations in anthropometric parameters, energy intake, appetite, lipid profile, insulin, and glucose levels.

| Socioeconomic profile
Majority of the children belonged to low-income backgrounds (monthly income from PKR. 5000 to 35,000). The fathers were mostly drivers, guards, and laborers. Similarly, most of the mothers were housewives. showed that majority of the children from both groups were living in separate families, urban areas and were using community tape water (81.25% in LNS and 87.50% in PLACEBO) ( Table 2).

| Blood parameters analysis
The levels of total cholesterol (TC) (mg/dl) (p < .01) and fasting glucose (mg/dl) (p < .01) were significantly high in the LNS group after 4 weeks of supplementation as compared to the PLACEBO group (Table 4).

| EI on first trial day
A significant increase in EI (p < .001), fats (p < .001), proteins (p < .001), and carbohydrates (p < .01) was observed in the LNS group. However, during breakfast and lunch, the total EI (p < .05) and carbohydrates intake (p < .01) was significantly lower in LNS.
Similarly, when the sum of energy intake during breakfast and lunch was analyzed, energy intake (p < .01) and intake of proteins (p < .05), fats (p < .05), and carbohydrates (p < .01) of LNS was found to be significantly lower than the PLACEBO group. Also, a significant decrease was observed in only carbohydrates (p < .01) in the LNS group when the total EI was combined (Table 5).

| EI on second trial day
After 4 weeks of supplementation, the energy intake (p < .001), proteins (p < .001), fats (p < .001) and carbohydrate (p < .01) was significantly high in the LNS group after provision of supplement. During breakfast (p < .05) and lunch (p < .001), only CHO intake was significantly lower in the LNS group. Also, a significant increase was found only in fats intake (p < .05) in the LNS group when total energy intake was calculated (Table 5).

| Multiple pass 24-hour dietary recalls measurements
No significant difference was observed in energy intake and the macronutrient intake between LNS and PLACEBO groups before the 1st trial day. However, after 3 days of the first trial day, the total energy, proteins, and fats intake were significantly raised in the LNS group (p < .001) as compared to the PLACEBO group. Similarly, after 15 days of the first trial day, again the energy (p < .001), proteins (p < .01), and fats (p < .001) intake were significantly higher in the LNS group in comparison to the PLACEBO group (p < .001) (Table S1).

| Appetite analysis
Appetite analysis showed that all appetite measures were not significantly different between the LNS group and the PLACEBO group.
However, during pre-lunch period (120-240 min) on first trial day, a significant increase (p < .01) was observed in summary response of satiety and fullness in the PLACEBO group as compared to the LNS group (Table S2, Figure 3b,c).
Similarly, on second trial day again during pre-lunch period (120-240 min), response of fullness was found significantly higher (p < .05) in the LNS group than in the PLACEBO group (Table S3, Figure 4c).

| Hedonic scale analysis
Most participants liked the taste of supplements. Only 6.25% of children slightly disliked the taste of LNS and PLACEBO from both the groups (Table S4).

| DISCUSS ION
High prevalence of childhood malnutrition in developing countries has significantly raised the overall mortality rate and disease burden (Smith et al., 2003). Over time, different interventional/   On first trial day, a significant decrease in energy intake during breakfast was observed in the LNS group after provision of supplement. Similar energy suppression was found during breakfast and dinner in other studies after the intake of supplement .

TA B L E 3 Comparison of anthropometric measurements between first and second trial days of the LNS group and the PLACEBO group
However, prolonged energy suppression was observed in our study which extended up to lunch in contrast to other two studies . Similar prolonged energy suppression after the use of LNS was observed in a recent study on malnourished children (Nawab et al., 2022) which shows that energy is compensated due to intake of high caloric supplement. On second trial day, the total energy intake was higher in the LNS group than in the PLACEBO group during breakfast and lunch. Similarly, no energy suppression is reported previously in other studies Nawab et al., 2022). This suggests that children can accommodate the feeding of supplement if it is given on daily basis and energy consumption is not reduced.
We also checked the habitual EI of participants using Multiplepass 24-hour dietary recalls 3 days before the first trial, 3 days after the first trial and then 15 days after the first trial day. Energy intake at the baseline showed no significant difference however, a significant increase was found in the LNS group in comparison to the PLACEBO group (p < .001) after three and 15 days of LNS. Similar results were observed in previous studies where supplementation caused significant increase in energy intake of participants Huynh et al., 2015;Parsons et al., 2017;Samadi et al., 2016).
In this study, we also found that the effect of the extra energy (535 kcal/day) provided by supplement on weight gain of participants was quite noticeable after 4 weeks of supplementation; however, it was much lower than expected. The average increase in the weight of LNS participants was 0.6 kg instead of expected 2 kg. It can be explained in terms of energy compensation due to supplement provision. Similar results were found in some other studies on undernourished children where the observed weight gain was lower than predicted values due to lowered habitual energy intake after using supplementation on daily basis (Fatima et al., 2018;Lazzerini et al., 2013;Lenters et al., 2013). Appetite assessment revealed suppression in breakfast in LNS but the appetite was not affected suggesting there was a short-term appetite suppression. On first trial day of our study, the PLACEBO group felt much satisfied and full as compared to the LNS group in pre-lunch period. This is also reported previously (Dossa et al., 2001(Dossa et al., , 2002. One of the reason for this short-term suppression could be the limited time span of supplement administration, breakfast, and lunch. This sort of suppression which was short-lived was also observed in underweight females when supplement was taken in the morning . Similarly, on last day of trial, appetite was not affected during pre-breakfast period; however, a significant rise was observed in fullness score of the LNS group during pre-lunch period. This indicates adaptation to the supplementation however, the short-term effect on appetite was still preserved. These results are in con-  Fabiansen et al., 2017). A significant improvement in weight gain of MAM children (Collins, 2004) and malnourished gastrointestinal patients was observed after the use of supplementation (Kennedy et al., 2015). Likewise, previous literature shows that use of LNS significantly improved BMI as well as MUAC in malnourished children (Dewey et al., 2021;Fabiansen et al., 2017). No change in the height was observed after 4 weeks of supplementation as it was for short duration. Similar to our findings short-term supplementation did not TA B L E 4 Comparison of lipid parameters, insulin, and glucose between first and second trial days of the LNS group and the PLACEBO group affect height or skin fold thickness to a significant level in malnourished adults and children (World Health Organization, 2007).
Glucose and insulin both play important roles in appetite regulation (Woods et al., 2006). In our study, a significant increase in baseline glucose levels was observed after 4 weeks of supplementation in the LNS group. The same was observed by Fatima et al.  in lean women after the use of high-energy nutritional supplement drinks (HENSDs). However, in contrast to our results, a study found no significant difference in glucose levels after using supplements for 15 days (Althuis et al., 2002).
We found no significant difference in insulin levels after LNS use.
Similar to our results, another study on healthy individuals found no significant difference in insulin levels after the administration of supplements (Althuis et al., 2002). However, in contrast to our results, insulin levels significantly raised in a trial performed on thin women after using HENSDs . These findings suggest that the way of metabolization of insulin might vary between women and children.
We compared the data on lipid profile before and after taking supplementation for 4 weeks. No significant difference was found in the PLACEBO group among lipid profile parameters on day 1st and 31st. However, we observed a significant increase in total cholesterol (TC) levels in the LNS group. In contrast to our study, a significant decrease in TC and HDL levels was observed after supplement use by individuals from different ethnic groups (Dodin et al., 2005). The reason for significantly higher levels of TC in our study might be related to ethnicity as lipid profile is multifactorial (Tejada et al., 1968). Furthermore, we found significant increase in fat content in the LNS group. The LNS used in our study also contains high-fat content (Table 1). Similarly, high TC levels were found in previous studies, where high-fat diet was used (Enos et al., 2013;Lorenzen & Astrup, 2011).
Strengths of this study were that EI data were analyzed by three researchers individually to enhance the accuracy of results.
Similarly, habitual EI was obtained using Multiple pass 24-hour dietary recalls to reduce misreporting biasness. Also, to maintain good compliance, supplements were provided on daily basis and empty sachets were collected by the main researchers on the same day. Limitation of this study was that mechanism of appetite suppression and appetite hormones like leptin and ghrelin could not be checked.
After our investigation, we recommend long-term studies to evaluate the effects of appetite hormones and appetite pattern in malnourished and healthy children. We also recommend nutritional counseling along with nutritional programs which might prove to be an effective strategy for tackling nutritional problems in children.

| CON CLUS ION
LNS causes improvement in nutritional outcomes of mild-tomoderate underweight children. It has proven to be effective in increasing the overall energy intake but it has also reduced the regular dietary intake of children due to which the observed weight gain was less than expected. Moreover, the use of LNS only for 1 month induces hyperglycemia and hyperlipidemia.

ACK N OWLED G M ENTS
We are grateful to all the participants and their parents for their participation in the study trial. Also, thanks to the co-researchers (Fouzia Nawab and Akhlaq Ahmad) for their countless support.

FU N D I N G I N FO R M ATI O N
The current research was partially funded to the research scholar Aqsa Zubair (MPhil scholar) by the Organization of Research

Innovation and commercialization (ORIC) of Khyber Medical
University.

DATA AVA I L A B I L I T Y S TAT E M E N T
Available from the corresponding author on reasonable request.

CO N FLI C T O F I NTE R E S T S
The authors declared no conflict of interests. All authors approved the final manuscript.